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WO2001058494A2 - Matieres et procedes destines au traitement de troubles oculaires - Google Patents

Matieres et procedes destines au traitement de troubles oculaires Download PDF

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Publication number
WO2001058494A2
WO2001058494A2 PCT/US2001/004203 US0104203W WO0158494A2 WO 2001058494 A2 WO2001058494 A2 WO 2001058494A2 US 0104203 W US0104203 W US 0104203W WO 0158494 A2 WO0158494 A2 WO 0158494A2
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid sequence
vector
sequence encoding
ocular
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PCT/US2001/004203
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English (en)
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WO2001058494A3 (fr
Inventor
Imre Kovesdi
Douglas E. Brough
Duncan L. Mcvey
Lisa Wei
Original Assignee
Genvec, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/599,997 external-priority patent/US6821775B1/en
Application filed by Genvec, Inc. filed Critical Genvec, Inc.
Priority to CA002398571A priority Critical patent/CA2398571A1/fr
Priority to EP01909022A priority patent/EP1253948A2/fr
Priority to JP2001557602A priority patent/JP2003522160A/ja
Priority to AU36818/01A priority patent/AU780634C/en
Publication of WO2001058494A2 publication Critical patent/WO2001058494A2/fr
Publication of WO2001058494A3 publication Critical patent/WO2001058494A3/fr
Priority to US10/211,701 priority patent/US20030045498A1/en
Priority to AU2005202935A priority patent/AU2005202935A1/en
Priority to US11/610,364 priority patent/US20070098692A1/en
Priority to US12/237,036 priority patent/US20090018100A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a method of prophylactically or therapeutically treating an ocular disorder as well as materials useful for treating an ocular disorder.
  • Nision loss affects virtually all people regardless of age, race, economic or social status, or geographical location. Ocular-related disorders, while often not life threatening, necessitate life-style changes that jeopardize the independence of the afflicted individual.
  • Nision impairment can result from most all ocular disorders, including diabetic retinopathies, proliferative retinopathies, retinal detachment, toxic retinopathies, retinal vascular diseases, retinal degenerations, vascular anomalies, age- related macular degeneration and other acquired disorders, infectious diseases, inflammatory diseases, ocular ischemia, pregnancy-related disorders, retinal tumors, choroidal tumors, choroidal disorders, vitreous disorders, trauma, cataract complications, dry eye, and inflammatory optic neuropathies.
  • ocular disorders including diabetic retinopathies, proliferative retinopathies, retinal detachment, toxic retinopathies, retinal vascular diseases, retinal degenerations, vascular anomalies, age- related macular degeneration and other acquired disorders, infectious diseases, inflammatory diseases, ocular ischemia, pregnancy-related disorders, retinal tumors, choroidal tumors, choroidal disorders, vitreous disorders, trauma, cataract complications, dry eye, and inflammatory optic neuropathies
  • Ocular-related diseases comprising a neovascularization aspect are many and include, for example, exudative age-related macular degeneration, diabetic retinopathy, corneal neovascularization, choroidal neovascularization, neovascular glaucoma, cyclitis, Hippel- Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated neovascularization, and the like.
  • the retina is a delicate ocular membrane on which images are received. Near the center of the retina is the macula lutea, an oval area of retinal tissue where visual sense is most acute. The retina is most delicate at the fovea centralis, the central depression located in the center of the macula. Damage of the retina, i.e., retinal detachment, retinal tears, or retinal degeneration, is directly connected to vision loss. While a common cause of retinal detachment, retinal tears, and retinal degeneration is abnormal, i.e., uncontrolled, vascularization of various ocular tissues, this is not always the case.
  • Atrophic complications associated with age-related macular degeneration, nonproliferative diabetic retinopathy, and inflammatory ocular damage are not associated with neovascularization, but can result in severe vision loss if not treated.
  • Disorders associated with both neovascular and atrophic components, such as age-related macular degeneration and diabetic retinopathy, are particularly difficult to treat due to the emergence of a wide variety of complications.
  • Age-related macular degeneration is the leading cause of blindness in patients over 65 years of age. As the elderly population of the world increases, the incidence of age-related macular degeneration is expected to increase dramatically, reaching a predicted 7.5 million cases in the United States alone by the year 2030 (Hyman et al., Am. J. Epidemiol., 118, 213-227 (1983)).
  • Age-related macular degeneration (AMD) is a progressive, degenerative disorder of the eye resulting initially in loss of visual acuity. Complications arising with advanced age-related macular degeneration include atrophic and exudative complications. Atrophic complications stem from retinal pigment epithelial cell loss resulting in atrophy of the retinal pigment epithelium (RPE).
  • Exudative complications include disciform scars (i.e., scarring involving fibrous elements) and neovascularization. Severe vision loss occurs as neovascularization or atrophy disturbs the foveal center (Bressler et al., Ophthalmology, 102, 1206-1211 (1995)). Ultimately, blindness from age- related macular degeneration stems from degeneration of the RPE and the subsequent death of photoreceptors.
  • diabetic retinopathy is subdivided into a nonproliferative stage, which typically occurs first, and a proliferative stage.
  • Nision loss associated with nonproliferative diabetic retinopathy stems from retinal edema, in particular diabetic macular edema, resulting from vascular leakage.
  • Focal and diffuse vascular leakage occurs as a result of microvascular abnormalities, intraretinal microaneurysms, capillary closure, and retinal hemorrhages. Prolonged periods of vascular leakage ultimately lead to thickening of the basement membrane and formation of soft and hard exudates.
  • Nonproliferative diabetic retinopathy is also characterized by loss of retinal pericytes.
  • the proliferative stage of diabetic retinopathy is characterized by neovascularization and fibrovascular growth (i.e., scarring involving glial and fibrous elements) from the retina or optic nerve over the inner surface of the retina or disc or into the vitreous cavity.
  • Retinal neovascularization is the leading cause of vision loss associated with proliferative diabetic retinopathy.
  • Laser photocoagulation involves administering laser burns to various areas of the eye and is used in the treatment of many neovascularization-linked disorders. For example, focal macular photocoagulation is used to treat areas of vascular leakage in the macula (Murphy, Amer.
  • neovascularization in particular, advanced proliferative retinopathy, is commonly treated with scatter or panretinal photocoagulation.
  • laser treatment may cause permanent blind spots corresponding to the treated areas.
  • Laser treatment may also cause persistent or recurrent hemorrhage, increase the risk of retinal detachment, or induce neovascularization or fibrosis.
  • age-related macular degeneration many patients eventually experience severe vision loss in spite of treatment.
  • Other treatment options for ocular- related disorders include thermotherapy, radiation therapy, surgery, e.g., macular translocation, removal of excess ocular tissue, and the like.
  • all available treatment options have limited therapeutic effect, require repeated, costly procedures, and/or are associated with dangerous side-effects.
  • the present invention provides materials and methods for prophylactically and therapeutically treating ocular-related disorders, including treatment of nonproliferative complications and proliferative complications.
  • the present invention provides a method for the prophylactic or therapeutic treatment of ocular-related disorders.
  • the present invention provides a method of prophylactically or therapeutically treating an animal for at least one ocular-related disorder, such as ocular neovascularization and age-related macular degeneration.
  • the method comprises contacting an ocular cell with (a) an expression vector comprising a nucleic acid sequence encoding an inhibitor of angiogenesis and the same or different nucleic acid sequence encoding a neurotrophic agent, or (b) different expression vectors, each comprising a nucleic acid sequence encoding an inhibitor of angiogenesis and/or a neurotrophic agent.
  • the nucleic acid sequence encoding the inhibitor of angiogenesis and/or the nucleic acid sequence encoding the neurotrophic agent is (are) expressed, thereby resulting in the production of the inhibitor of angiogenesis and/or the neurotrophic agent to prophylactically or therapeutically treat the animal for an ocular-related disorder.
  • the nucleic acid sequence encoding the inhibitor of angiogenesis and the nucleic acid sequence encoding the neurotrophic agent are the same nucleic acid sequence. More preferably, the nucleic acid sequence encodes a factor comprising both anti-angiogenic and neurotrophic activity. Most preferably, the factor is PEDF.
  • the present invention provides a viral vector comprising a nucleic acid sequence encoding pigment epithelium-derived factor (PEDF) or a therapeutic fragment thereof.
  • the nucleic acid sequence is operably linked to regulatory sequences necessary for expression of PEDF or a therapeutic fragment thereof.
  • the viral vector is an adenoviral vector or an adeno-associated viral vector.
  • the viral vector further comprises one or more additional nucleic acid sequences encoding therapeutic substances other than PEDF or a therapeutic fragment thereof.
  • the present invention is directed to methods of prophylactically or therapeutically treating an animal, preferably a human, for at least one ocular-related disorder.
  • the present invention also provides materials for treating ocular-related disorders.
  • Ocular-related disorders appropriate for treatment using the present inventive materials and methods include, but are not limited to, diabetic retinopathies, proliferative retinopathies, retinopathy of prematurity, retinal vascular diseases, vascular anomalies, age- related macular degeneration and other acquired disorders, endophthalmitis, infectious diseases, inflammatory diseases, AIDS-related disorders, ocular ischemia syndrome, pregnancy-related disorders, peripheral retinal degenerations, retinal degenerations, toxic retinopathies, cataracts, retinal tumors, corneal neovascularization, choroidal tumors, choroidal disorders, choroidal neovascularization, neovascular glaucoma, vitreous disorders, retinal detachment and proliferative vitreoretinopathy, cyclitis, non-penetrating trauma, penetrating trauma, post-cataract complications, Hippel-Lindau Disease, dry eye, inflammatory optic neuropathies, macular edema, ptery
  • the present invention provides a method of prophylactically or therapeutically treating an animal for at least one ocular-related disorder, such as ocular neovascularization.
  • the method comprises contacting an ocular cell with an expression vector comprising a nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic agent.
  • the method comprises contacting an ocular cell with an expression vector comprising a nucleic acid sequence encoding an inhibitor of angiogenesis and the same or different nucleic acid sequence encoding a neurotrophic agent.
  • the nucleic acid sequence encodes at least one inhibitor of angiogenesis and at least one neurotrophic agent.
  • the ocular neovascularization treated by the present inventive method can be neovascularization of the choroid.
  • the choroid is a thin, vascular membrane located under the retina.
  • Abnormal neovascularization of the choroid results from, for example, photocoagulation, anterior ischemic optic neuropathy, Best's disease, choroidal hemangioma, metallic intraocular foreign body, choroidal nonperfusion, choroidal osteomas, choroidal rupture, bacterial endocarditis, choroideremia, chronic retinal detachment, drusen, deposit of metabolic waste material, endogenous Candida endophthalmitis, neovascularization at ora serrata, operating microscope burn, punctate inner choroidopathy, radiation retinopathy, retinal cryoinjury, retinitis pigmentosa, retinochoroidal coloboma, rubella, subretinal fluid drainage, tilted disc syndrome, Taxoplasma retinochoroiditis, tuber
  • Neovascularization of the cornea is also appropriate for treatment by the method of the present invention.
  • the cornea is a projecting, transparent section of the fibrous tunic, the outer most layer of the eye.
  • the outermost layer of the cornea contacts the conjunctiva, while the innermost layer comprises the endothelium of the anterior chamber.
  • Corneal neovascularization stems from, for example, ocular injury, surgery, infection, improper wearing of contact lenses, and diseases such as, for example, corneal dystrophies.
  • the ocular neovascularization is preferably neovascularization of the retina. Retinal neovascularization is an indication associated with numerous ocular diseases and disorders, many of which are named above.
  • the neovascularization of the retina treated in accordance with the present inventive method is associated with diabetic retinopathy.
  • Common causes of retinal neovascularization include ischemia, viral infection, and retinal damage.
  • Neovascularization of the retina can lead to macular edema, subretinal discoloration, scarring, and the like.
  • Complications associated with retina neovascularization stem from breakage and leakage of newly formed blood vessels. Vision is impaired as blood fills the vitreous cavity and is not efficiently removed. Not only is the passage of light impeded, but an inflammatory response to the excess blood and metabolites can cause further damage to ocular tissue.
  • the present invention also provides a method for prophylactically or therapeutically treating an animal for age-related macular degeneration.
  • the method comprises contacting an ocular cell associated with age-related macular degeneration with an expression vector comprising a nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic factor.
  • the expression vector comprises a nucleic acid sequence encoding an inhibitor of angiogenesis and a nucleic acid sequence encoding a neurotrophic agent.
  • the nucleic acid sequence encoding the inhibitor of angiogenesis and the nucleic acid sequence encoding the neurotrophic agent are the same nucleic acid sequence.
  • the age-related macular degeneration is associated with at least one exudative complication. Exudative complications include, for example, disciform scars (i.e., scarring involving fibrous elements) and neovascularization.
  • the age-related macular degeneration is associated with at least one atrophic complication.
  • Atrophic complications include, for instance, the formation of drusen and basal laminar deposits, irregularity of retinal pigmentation, and accumulation of lipofuscin granules.
  • prophylactic is meant the protection, in whole or in part, against ocular- related disorders, in particular ocular neovascularization or age-related macular degeneration.
  • therapeutic is meant the amelioration of the ocular-related disorder, itself, and the protection, in whole or in part, against further ocular-related disease, in particular ocular neovascularization or age-related macular degeneration.
  • a therapeutic agent can be directly applied to affected areas without the harmful side effects of presently employed therapies.
  • the present inventive methods are useful in the treatment of both acute and persistent, progressive ocular-related disorders.
  • the expression vector comprising a nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic factor can be administered using a single or multiple applications within a short time period.
  • numerous applications of the expression vector may be necessary to realize a therapeutic effect.
  • suitable expression vectors include, for instance, plasmids, plasmid-liposome complexes, and viral vectors, e.g., parvoviral-based vectors (i.e., adeno-associated virus ( ⁇ AV)-based vectors), retroviral vectors, herpes simplex virus (HSN)-based vectors, AAN- adenoviral chimeric vectors, and adeno virus-based vectors.
  • parvoviral-based vectors i.e., adeno-associated virus ( ⁇ AV)-based vectors
  • retroviral vectors i.e., adeno-associated virus ( ⁇ AV)-based vectors
  • retroviral vectors i.e., adeno-associated virus ( ⁇ AV)-based vectors
  • HSN herpes simplex virus
  • AAN- adenoviral chimeric vectors adeno virus-based vectors.
  • Plasmids genetically engineered circular double-stranded DNA molecules, can be designed to contain an expression cassette for delivery of the nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic factor to an ocular cell.
  • Plasmids are often used for short-term expression. However, a plasmid construct can be modified to obtain prolonged expression.
  • the expression vector can be a plasmid comprising native parvovirus ITRs to obtain prolonged and substantial expression of at least one inhibitor of angiogenesis and/or at least one neurotrophic factor. While plasmids are suitable for use in the present inventive methods, preferably the expression vector is a viral vector.
  • AAN vectors are viral vectors of particular interest for use in gene therapy protocols.
  • AAN is a D ⁇ A virus, which is not known to cause human disease.
  • AAN requires co-infection with a helper virus (i.e., an adeno virus or a herpes virus), or expression of helper genes, for efficient replication.
  • helper virus i.e., an adeno virus or a herpes virus
  • AAN vectors used for administration of a therapeutic nucleic acid have approximately 96% of the parental genome deleted, such that only the terminal repeats (ITRs), which contain recognition signals for D ⁇ A replication and packaging, remain. This eliminates immunologic or toxic side effects due to expression of viral genes.
  • delivering the AAN rep protein enables integration of the AAN vector comprising AAN ITRs into a specific region of genome, if desired.
  • Retrovirus is an R ⁇ A virus capable of infecting a wide variety of host cells. Upon infection, the retroviral genome integrates into the genome of its host cell and is replicated along with host cell D ⁇ A, thereby constantly producing viral R ⁇ A and any nucleic acid sequence incorporated into the retroviral genome.
  • retroviral vectors can additionally be manipulated to render the virus replication-incompetent.
  • retroviral vectors are thought to be particularly useful for stable gene transfer in vivo.
  • Lentiviral vectors such as HIN -based vectors, are exemplary of retroviral vectors used for gene delivery.
  • HIN-based vectors are known to incorporate their passenger genes into non-dividing cells and, therefore, can be of use in treating atrophic forms of ocular-related disease.
  • HSN-based viral vectors are suitable for use as an expression vector to introduce nucleic acids into ocular cells.
  • the mature HSN virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded D ⁇ A molecule that is 152 kb.
  • Most replication-deficient HSN vectors contain a deletion to remove one or more intermediate-early genes to prevent replication.
  • Advantages of the herpes vector are its ability to enter a latent stage that can result in long-term D ⁇ A expression, and its large viral D ⁇ A genome that can accommodate exogenous D ⁇ A up to 25 kb. Of course, this ability is also a disadvantage in terms of short-term treatment regimens.
  • HSN-based vectors appropriate for use in the present inventive methods, see, for example, U.S. Patents 5,837,532; 5,846,782; 5,849,572; and 5,804,413 and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583.
  • Adenovirus is a 36 kb double-stranded D ⁇ A virus that efficiently transfers D ⁇ A in vivo to a variety of different target cell types.
  • the virus is preferably made replication deficient by deleting select genes required for viral replication.
  • the expendable E3 region is also frequently deleted to allow additional room for a larger D ⁇ A insert.
  • the vector can be produced in high titers and can efficiently transfer D ⁇ A to replicating and non-replicating cells.
  • the newly transferred genetic information remains epi-chromosomal, thus eliminating the risks of random insertional mutagenesis and permanent alteration of the genotype of the target cell.
  • the integrative properties of AAN can be conferred to adenovirus by constructing an AAN -Ad chimeric vector.
  • the AAN ITRs and nucleic acid encoding the Rep protein incorporated into an adenoviral vector enables the adenoviral vector to integrate into a mammalian cell genome. Therefore, AAN-Ad chimeric vectors are an interesting option for use in the present invention.
  • the expression vector of the present inventive methods is a viral vector; more preferably, the expression vector is an adenoviral vector.
  • the adenoviral vector can be derived from any serotype of adenovirus.
  • Adenoviral stocks that can be employed as a source of adenovirus can be amplified from the adenoviral serotypes 1 through 51, which are currently available from the American Type Culture Collection (ATCC, Manassas, NA), or from any other serotype of adenovirus available from any other source.
  • an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviral serotype.
  • an adenovirus is of serotype 2, 5 or 9.
  • non-group C adenoviruses can be used to prepare replication- deficient adenoviral gene transfer vectors for delivery of anti-angiogenic factors and/or neurotrophic factors to ocular cells.
  • Preferred adenoviruses used in the construction of non- group C adenoviral gene transfer vectors include Ad 12 (group A), Ad7 (group B), Ad30 and Ad36 (group D), Ad4 (group E), and Ad41 (group F).
  • Non-group C adenoviral vectors, methods of producing non-group C adenoviral vectors, and methods of using non-group C adenoviral vectors are disclosed in, for example, U.S.
  • the adenoviral vector is preferably deficient in at least one gene function required for viral replication, thereby resulting in a "replication-deficient" adenoviral vector.
  • the adenoviral vector is deficient in at least one essential gene function of the El region of the adenoviral genome required for viral replication.
  • the recombinant adenovirus can also have a mutation in the major late promoter (MLP).
  • the mutation in the MLP can be in any of the MLP control elements such that it alters the responsiveness of the promoter, as discussed in International Patent Application WO 00/00628. More preferably, the vector is deficient in at least one essential gene function of the El region and at least part of the E3 region (e.g., an Xba I deletion of the E3 region). With respect to the El region, the adenoviral vector can be deficient in at least part of the El a region and at least part of the Elb region. Preferably, the adenoviral vector is "multiply deficient," meaning that the adenoviral vector is deficient in one or more essential gene functions required for viral replication in each of two or more regions.
  • the aforementioned El-deficient or E1-, E3-deficient adenoviral vectors can be further deficient in at least one essential gene of the E4 region.
  • Adenoviral vectors deleted of the entire E4 region can elicit lower host immune responses.
  • the adenoviral vector lacks all or part of the El region and all or part of the E2 region.
  • adenoviral vectors lacking all or part of the El region, all or part of the E2 region, and all or part of the E3 region also are contemplated herein.
  • the adenoviral vector lacks all or part of the El region, all or part of the E2 region, all or part of the E3 region, and all or part of the E4 region.
  • Suitable replication- deficient adenoviral vectors are disclosed in U.S. Patents 5,851,806 and 5,994,106 and International Patent Applications WO 95/34671 and WO 97/21826.
  • suitable replication-deficient adenoviral vectors include those with at least a partial deletion of the El a region, at least a partial deletion of the Elb region, at least a partial deletion of the E2a region, and at least a partial deletion of the E3 region.
  • the replication- deficient adenoviral vector can have at least a partial deletion of the El region, at least a partial deletion of the E3 region, and at least a partial deletion of the E4 region.
  • multiply-deficient viral vectors are particularly useful in that such vectors can accept large inserts of exogenous DNA.
  • adenoviral amplicons an example of a multiply- deficient adenoviral vector which comprises only those genomic sequences required for packaging and replication of the viral genome, can accept inserts of approximately 36 kb.
  • the expression vector of the present inventive method is a multiply-deficient adenoviral vector lacking all or part of the El region, all or part of the E3 region, all or part of the E4 region, and, optionally, all or part of the E2 region.
  • an at least E4-def ⁇ cient adenoviral vector expresses a transgene at high levels for a limited amount of time in vivo and that persistence of expression of a transgene in an at least E4-def ⁇ cient adenoviral vector can be modulated through the action of a trans-acting factor, such as HSN ICPO, Ad pTP, CMN- IE2, CMN-IE86, HIN tat, HTLN-tax, HBN-X, AAN Rep 78, the cellular factor from the U205 osteosarcoma cell line that functions like HSN ICPO, or the cellular factor in PC 12 cells that is induced by nerve growth factor, among others.
  • a trans-acting factor such as HSN ICPO, Ad pTP, CMN- IE2, CMN-IE86, HIN tat, HTLN-tax, HBN-X, AAN Rep 78, the cellular factor from the U205 osteosarcoma cell line that
  • the multiply deficient adenoviral vector e.g., the at least E4-deficient adenoviral vector
  • the ocular cell is contacted with a second expression vector comprising a nucleic acid sequence encoding a trans-acting factor that modulates the persistence of expression of the nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic factor.
  • the nucleic acid sequence encoding the trans-acting factor does not encode an adenoviral E4 region gene product.
  • the trans-acting factor is the Herpes simplex infected cell polypeptide 0 (HSN ICPO).
  • the adenoviral vector's coat protein can be modified so as to decrease the adenoviral vector's ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in International Patent Application WO 98/40509. Such modifications are useful for long-term treatment of persistent ocular disorders.
  • the coat protein of a viral vector can be manipulated to alter the binding specificity or recognition of a virus for a viral receptor on a potential host cell.
  • adenovirus such manipulations can include deletion of regions of the fiber, penton, or hexon, insertions of various native or non-native ligands into portions of the coat protein, and the like.
  • Manipulation of the coat protein can broaden the range of cells infected by a viral vector or enable targeting of a viral vector to a specific cell type.
  • the expression vector is a viral vector comprising a chimeric coat protein (e.g., a fiber, hexon pIX, pllla, or penton protein), which differs from the wild-type (i.e., native) coat protein by the introduction of a normative amino acid sequence, preferably at or near the carboxyl terminus.
  • a normative amino acid sequence is inserted into or in place of an internal coat protein sequence.
  • the normative amino acid sequence can be inserted within the internal coat protein sequence or at the end of the internal coat protein sequence-
  • the resultant chimeric viral coat protein is able to direct entry into cells of the viral, i.e., adenoviral, vector comprising the coat protein that is more efficient than entry into cells of a vector that is identical except for comprising a wild-type viral coat protein rather than the chimeric viral coat protein.
  • the chimeric virus coat protein binds a novel endogenous binding site present on the cell surface that is not recognized, or is poorly recognized by a vector comprising a wild-type coat protein.
  • the virus preferably, the adenovirus, can bind to and enter numerous cell types which a virus comprising wild-type coat protein typically cannot enter or can enter with only a low efficiency.
  • the expression vector is a viral vector comprising a chimeric virus coat protein not selective for a specific type of eukaryotic cell.
  • the chimeric coat protein differs from the wild-type coat protein by an insertion of a normative amino acid sequence into or in place of an internal coat protein sequence.
  • the chimeric virus coat protein efficiently binds to a broader range of eukaryotic cells than a wild-type virus coat, such as described in International Patent Application WO 97/20051.
  • Specificity of binding of an adenovirus to a given cell can also be adjusted by use of an adenovirus comprising a short-shafted adenoviral fiber gene, as discussed in U.S. Patent 5,962,311.
  • Use of an adenovirus comprising a short-shafted adenoviral fiber gene reduces the level or efficiency of adenoviral fiber binding to its cell-surface receptor and increases adenoviral penton base binding to its cell-surface receptor, thereby increasing the specificity of binding of the adenovirus to a given cell.
  • an adenovirus comprising a short-shafted fiber enables targeting of the adenovirus to a desired cell-surface receptor by the introduction of a normative amino acid sequence either into the penton base or the fiber knob.
  • the ability of a viral vector to recognize a potential host cell can be modulated without genetic manipulation of the coat protein. For instance, complexing an adenovirus with a bispecific molecule comprising a penton base-binding domain and a domain that selectively binds a particular cell surface binding site enables one of ordinary skill in the art to target the vector to a particular cell type.
  • Suitable modifications to a viral vector are described in U.S. Patents 5,559,099; 5,731,190; 5,712,136; 5,770,442; 5,846,782; 5,926,311; 5,965,541; 6,057,155; 6,127,525; and 6,153,435 and International Patent Applications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO 98/07877, WO 98/54346, and WO 00/15823.
  • numerous expression vectors are available commercially. Construction of expression vectors is well understood in the art.
  • Adenoviral vectors can be constructed and/or purified using the methods set forth, for example, in U.S. Patent 5,965,358 and International Patent Applications WO 98/56937, WO 99/15686, and WO 99/54441.
  • Adeno-associated viral vectors can be constructed and/or purified using the methods set forth, for example, in U.S. Patent 4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).
  • expression vector for use in the present inventive method will depend on a variety of factors such as, for example, the host, immunogenicity of the vector, the desired duration of protein production, and the like. As each type of expression vector has distinct properties, a researcher has the freedom to tailor the present inventive method to any particular situation. Moreover, more than one type of expression vector can be used to deliver the nucleic acid sequence to the ocular cell.
  • the present invention provides a method of prophylactically or therapeutically treating an animal for at least one ocular- related disorder, wherein the method comprises contacting an ocular cell with different expression vectors, each comprising a nucleic acid sequence encoding an inhibitor of angiogenesis and/or a nucleic acid sequence encoding a neurotrophic agent.
  • the nucleic acid sequence encoding the inhibitor of angiogenesis and/or the nucleic acid sequence encoding the neurotrophic agent are expressed, thereby resulting in the production of the inhibitor of angiogenesis and/or the neurotrophic agent to prophylactically or therapeutically treat the animal for an ocular-related disorder.
  • At least two different types of expression vector are delivered to the ocular cell.
  • At least one expression vector can comprise a nucleic acid sequence encoding an inhibitor of angiogenesis.
  • at least one expression vector can comprise a nucleic acid sequence encoding a neurotrophic agent.
  • a mixture of expression vectors, some comprising the coding sequence for an inhibitor of angiogenesis and some comprising the coding sequence for a neurotrophic agent can be administered.
  • at least one expression vector comprises the nucleic acid sequence encoding the inhibitor of angiogenesis and the nucleic acid sequence encoding the neurotrophic agent.
  • the nucleic acid sequence encoding the inhibitor of angiogenesis and the nucleic acid sequence encoding the neurotrophic agent are the same nucleic acid.
  • the inhibitor of angiogenesis and the neurotrophic agent are a single factor.
  • the ocular cell is contacted with an adenoviral vector and an adeno-associated viral vector.
  • the method comprises contacting an ocular cell associated with age-related macular degeneration with an expression vector comprising a nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic factor.
  • the ocular cell is contacted with at least two different types of expression vector, each expression vector comprising a nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic factor.
  • age-related macular degeneration commonly afflicts the elderly, preferably the expression vector is administered to an animal, i.e., a human, at least 55 years old.
  • Age-related macular degeneration is a complex disease associated with a wide variety of complications affecting a number of ocular tissues.
  • Ocular cells associated with age-related macular degeneration include, but are not limited to, cells of neural origin, cells of all layers of the retina, especially retinal pigment epithelial cells, glial cells, and pericytes.
  • Other ocular cells that are suitable for use in the method of the present invention include, for example, endothelial cells, iris epithelial cells, corneal cells, ciliary epithelial cells, Mueller cells, astrocytes, and cells of the trabecular meshwork.
  • the trabecular meshwork is associated with the passage for fluid drainage out of the eye.
  • Other cells linked to various ocular-related diseases include, for example, fibroblasts and vascular endothelial cells.
  • the expression vector is administered to an area of vascular leakage.
  • the nucleic acid sequence is operably linked to regulatory sequences necessary for expression, i.e., a promoter.
  • a "promoter” is a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis.
  • a nucleic acid sequence is "operably linked" to a promoter when the promoter is capable of directing transcription of that nucleic acid sequence.
  • a promoter can be native or non- native to the nucleic acid sequence to which it is operably linked.
  • Any promoter i.e., whether isolated from nature or produced by recombinant DNA or synthetic techniques
  • the promoter preferably is capable of directing transcription in a eukaryotic (desirably mammalian) cell.
  • the functioning of the promoter can be altered by the presence of one or more enhancers and/or silencers present on the vector.
  • Enhanccers are cis-acting elements of DNA that stimulate or inhibit transcription of adjacent genes.
  • Enhancers differ from DNA-binding sites for sequence-specific DNA binding proteins found only in the promoter (which also are termed “promoter elements”) in that enhancers can function in either orientation, and over distances of up to several kilobase pairs (kb), even from a position downstream of a transcribed region.
  • promoter elements DNA-binding sites for sequence-specific DNA binding proteins found only in the promoter (which also are termed “promoter elements”) in that enhancers can function in either orientation, and over distances of up to several kilobase pairs (kb), even from a position downstream of a transcribed region.
  • the TATA box directs RNA polymerase to begin transcribing approximately 30 base pairs (bp) downstream in mammalian systems.
  • the TATA box functions in conjunction with at least two other upstream sequences located about 40 bp and 110 bp upstream of the start of transcription.
  • a so-called "CCAAT box” serves as one of the two upstream sequences, and the other often is a GC-rich segment.
  • the CCAAT homology can reside on different strands of the DNA.
  • the upstream promoter element also can be a specialized signal such as one of those which have been described in the art and which appear to characterize a certain subset of genes.
  • the TATA box and the upstream sequences are each recognized by regulatory proteins which bind to these sites, and activate transcription by enabling RNA polymerase II to bind the DNA segment and properly initiate transcription.
  • base changes outside the TATA box and the upstream sequences have little effect on levels of transcription, base changes in either of these elements substantially lower transcription rates (see, e.g., Myers et al., Science, 229, 242-247 (1985); McKnight et al., Science, 217, 316-324 (1982)).
  • the position and orientation of these elements relative to one another, and to the start site, are important for the efficient transcription of some, but not all, coding sequences. For instance, some promoters function well in the absence of any TATA box.
  • promoter regions can vary in length and sequence and can further encompass one or more DNA binding sites for sequence-specific DNA binding proteins and/or an enhancer or silencer. Enhancers and/or silencers can similarly be present on a nucleic acid sequence outside of the promoter per se.
  • the present invention preferentially employs a viral promoter.
  • Suitable viral promoters include, for instance, cytomegalovirus (CMN) promoters, such as the CMN immediate-early promoter, promoters derived from human immunodeficiency virus (HIN), such as the HIN long terminal repeat promoter, Rous sarcoma virus (RSN) promoters, such as the RSN long terminal repeat, mouse mammary tumor virus (MMTN) promoters, HSN promoters, such as the Lap2 promoter or the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad.
  • CPN cytomegalovirus
  • HIN human immunodeficiency virus
  • RSN Rous sarcoma virus
  • MMTN mouse mammary tumor virus
  • HSN promoters such as the Lap2 promoter or the herpes thymidine kinase promoter
  • promoters derived from SN40 or Epstein Barr virus promoters derived from SN40 or Epstein Barr virus, an adeno-associated viral promoter, such as the p5 promoter, and the like.
  • the viral promoter is an adenoviral promoter, such as the Ad2 or Ad5 major late promoter and tripartite leader, a CMN promoter, or an RSN promoter.
  • the promoter can be an inducible promoter, i.e., a promoter that is up- and/or down-regulated in response to appropriate signals.
  • the regulatory sequences can comprise a hypoxia driven promoter, which is active when the ocular neovascularization or age-related macular degeneration is associated with hypoxia.
  • suitable inducible promoter systems include, but are not limited to, the IL-8 promoter, the metallothionine inducible promoter system, the bacterial lacZYA expression system, the tetracycline expression system, and the T7 polymerase system.
  • promoters that are selectively activated at different developmental stages can be employed.
  • the promoter sequence that regulates expression of the nucleic acid sequence can contain at least one heterologous regulatory sequence responsive to regulation by an exogenous agent.
  • the regulatory sequences are preferably responsive to exogenous agents such as, but not limited to, drugs, hormones, or other gene products.
  • the regulatory sequences, e.g., promoter preferably are responsive to glucocorticoid receptor-hormone complexes, which, in turn, enhance the level of transcription of a therapeutic peptide or a therapeutic fragment thereof.
  • the regulatory sequences comprise a tissue-specific promoter, i.e., a promoter that is preferentially activated in a given tissue and results in expression of a gene product in the tissue where activated.
  • a typically used tissue-specific promoter is a myocyte-specific promoter.
  • a promoter exemplary of a myocyte-specific promoter is the myosin light-chain 1 A promoter.
  • a tissue specific promoter for use in the present inventive vector can be chosen by the ordinarily skilled artisan based upon the target tissue or cell- type.
  • Preferred tissue-specific promoters for use in the present inventive methods are specific to ocular tissue, such as a rhodopsin promoter. Examples of rhodopsin promoters include, but are not limited to, a GNAT cone- transducing alpha-subunit gene promoter or an interphotoreceptor retinoid binding protein promoter.
  • each promoter drives transcription, and, therefore, protein expression, differently with respect to time and amount of protein produced.
  • the CMN promoter is characterized as having peak activity shortly after transduction, i.e., about 24 hours after transduction, then quickly tapering off.
  • the RSN promoter's activity increases gradually, reaching peak activity several days after transduction, and maintains a high level of activity for several weeks.
  • sustained expression driven by an RSN promoter has been observed in all cell types studied, including, for instance, liver cells, lung cells, spleen cells, diaphragm cells, skeletal muscle cells, and cardiac muscle cells.
  • a promoter can be selected for use in the methods of the present invention by matching its particular pattern of activity with the desired pattern and level of expression of at least one inhibitor of angiogenesis and/or at least one neurotrophic factor.
  • a hybrid promoter can be constructed which combines the desirable aspects of multiple promoters. For example, a CMN-RSN hybrid promoter combining the CMN promoter's initial rush of activity with the RSN promoter's high maintenance level of activity would be especially preferred for use in many embodiments of the present inventive method. It is also possible to select a promoter with an expression profile that can be manipulated by an investigator.
  • the expression vector comprises a nucleic acid encoding a cis- acting factor, wherein the cis-acting factor modulates the expression of the nucleic acid sequence.
  • the cis-acting factor comprises matrix attachment region (MAR) sequences (e.g., immunoglobulin heavy chain (Jenunwin et al., Nature, 385(16), 269 (1997)), apolipoprotein B, or locus control region (LCR) sequences, among others.
  • MAR sequences have been characterized as D ⁇ A sequences that associate with the nuclear matrix after a combination of nuclease digestion and extraction (Bode et al., Science, 255(5041), 195-197 (1992)).
  • MAR sequences are often associated with enhancer-type regulatory regions and, when integrated into genomic D ⁇ A, MAR sequences augment transcriptional activity of adjacent nucleotide sequences. It has been postulated that MAR sequences play a role in controlling the topological state of chromatin structures, thereby facilitating the formation of transcriptionally-active complexes. Similarly, it is believed LCR sequences function to establish and/or maintain domains permissive for transcription. Many LCR sequences give tissue specific expression of associated nucleic acid sequences. Addition of MAR or LCR sequences to the expression vector can further enhance expression of at least one inhibitor of angiogenesis and/or at least one neurotrophic factor.
  • nucleic acid sequences, selectable markers, and the like, located on an expression vector according to the present invention can be present as part of a cassette, either independently or coupled.
  • a "cassette” is a particular base sequence that possesses functions which facilitate subcloning and recovery of nucleic acid sequences (e.g., one or more restriction sites) or expression (e.g., polyadenylation or splice sites) of particular nucleic acid sequences.
  • nucleic acid sequences are well within the skill of the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989)). With respect to the expression of nucleic acid sequences according to the present invention, the ordinary skilled artisan is aware that different genetic signals and processing events control levels of nucleic acids and proteins/peptides in a cell, such as, for instance, transcription, mRNA translation, and post-transcriptional processing. Transcription of DNA into RNA requires a functional promoter, as described herein.
  • Protein expression is dependent on the level of RNA transcription that is regulated by DNA signals, and the levels of DNA template.
  • translation of mRNA requires, at the very least, an AUG initiation codon, which is usually located within 10 to 100 nucleotides of the 5' end of the message. Sequences flanking the AUG initiator codon have been shown to influence its recognition by eukaryotic ribosomes, with conformity to a perfect Kozak consensus sequence resulting in optimal translation (see, e.g., Kozak, J Molec. Biol, 196, 947-950 (1987)). Also, successful expression of an exogenous nucleic acid in a cell can require post-translational modification of a resultant protein.
  • production of a protein can be affected by the efficiency with which DNA (or RNA) is transcribed into mRNA, the efficiency with which mRNA is translated into protein, and the ability of the cell to carry out post-translational modification.
  • the nucleic acid sequence further comprises a polyadenylation site following the coding region of the nucleic acid sequence. Also, preferably all the proper transcription signals (and translation signals, where appropriate) will be correctly arranged such that the nucleic acid sequence will be properly expressed in the cells into which it is introduced. If desired, the nucleic acid sequence also can incorporate splice sites (i.e., splice acceptor and splice donor sites) to facilitate mRNA production.
  • splice sites i.e., splice acceptor and splice donor sites
  • nucleic acid sequence encodes a protein or peptide, which is a processed or secreted protein or acts intracellularly
  • nucleic acid sequence further comprises the appropriate sequences for processing, secretion, intracellular localization, and the like.
  • an especially preferred method of modulating expression of a nucleic acid sequence comprises addition of site-specific recombination sites on the expression vector. Contacting an expression vector comprising site-specific recombination sites with a recombinase will either up- or down-regulate transcription of a coding sequence, or simultaneously up-regulate transcription one coding sequence and down-regulate transcription of another, through the recombination event.
  • site-specific recombination to modulate transcription of a nucleic acid sequence is described in, for example, U.S. Patents 5,801,030 and 6,063,627 and International Patent Application WO 97/09439.
  • the expression vector of the present inventive method comprises a nucleic acid encoding an inhibitor of angiogenesis. More preferably, the nucleic acid sequence encodes multiple inhibitors of angiogenesis.
  • inhibitor of angiogenesis is meant any factor that prevents or ameliorates neovascularization.
  • complete prevention or amelioration of neovascularization is not required in order to realize a therapeutic effect. Therefore, the present inventive methods contemplate both partial and complete prevention and amelioration of angiogenesis.
  • An inhibitor of angiogenesis includes, for instance, an anti-angiogenic factor, an anti-sense molecule specific for an angiogenic factor, a ribozyme, a receptor for an angiogenic factor, and an antibody that binds a receptor for an angiogenic factor.
  • the anti-angiogenic factors contemplated for use in the present invention include pigment epithelium-derived factor, angiostatin, vasculostatin, endostatin, platelet factor 4, heparinase, interferons (e.g., INF ⁇ ), and the like.
  • any anti-angiogenic factor can be modified or truncated and retain anti- angiogenic activity.
  • active fragments of anti-angiogenic factors i.e., those fragments having biological activity sufficient to inhibit angiogenesis are also suitable for use in the present inventive methods.
  • An anti-sense molecule specific for an angiogenic factor should generally be substantially identical to at least a portion, preferably at least about 20 continuous nucleotides, of the nucleic acid encoding the angiogenic factor to be inhibited, but need not be identical.
  • the anti-sense nucleic acid molecule can be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the nucleic acid.
  • the introduced anti-sense nucleic acid molecule also need not be full-length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence.
  • anti-sense molecule need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective.
  • Antisense phosphorothiotac oligodeoxynucleotides is exemplary of an anti-sense molecule specific for an angiogenic factor.
  • Ribozymes can be designed that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered and is, thus, capable of recycling and cleaving other molecules, making it a true enzyme.
  • the inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
  • the design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature, 334, 585-591 (1988).
  • the ribozyme comprises at least about 20 continuous nucleotides complementary to the target sequence on each side of the active site of the ribozyme.
  • Receptors specific for angiogenic factors inhibit neovascularization by sequestering growth factors away from functional receptors capable of promoting a cellular response.
  • Fit and Flk receptors as well as NEGF-receptor chimeric proteins, compete with NEGF receptors on vascular endothelial cells to inhibit endothelial cell growth (Aiello, PNAS, 92, 10457 (1995)).
  • growth factor-specific antibodies and fragments thereof e.g., Fab, F(ab') 2 , and Fv
  • neutralize angiogenic factors or bind receptors for angiogenic factors are also contemplated.
  • the present invention also contemplates delivery of a nucleic acid sequence encoding at least one neurotrophic agent (or neurotrophic factor) to ocular cells or cells associated with age-related macular degeneration.
  • Neurotrophic factors are thought to be responsible for the maturation of developing neurons and for maintaining adult neurons.
  • Neurotrophic factors are divided into three subclasses: neuropoietic cytokines; neurotrophins; and the fibroblast growth factors.
  • Ciliary neurotrophic factor (CNTF) is exemplary of neuropoietic cytokines. CNTF promotes the survival of ciliary ganglionic neurons and supports certain neurons that are NGF-responsive.
  • Neurotrophins include, for example, brain-derived neurotrophic factor and nerve growth factor, perhaps the best characterized neurotrophic factor.
  • Other neurotrophic factors suitable for being encoded by the nucleic acid sequence of the present inventive methods include, for example, transforming growth factors, glial cell-line derived neurotrophic factor, neurotrophin 3, neurotrophin 4/5, and interleukin 1- ⁇ .
  • Neurotrophic factors associated with angiogenesis such as aFGF and bFGF, are less preferred.
  • the neurotrophic factor of the present inventive method can also be a neuronotrophic factor, e.g., a factor that enhances neuronal survival. It has been postulated that neurotrophic factors can actually reverse degradation of neurons.
  • the nucleic acid sequence of the present invention encodes both an inhibitor of angiogenesis and a neurotrophic factor. More preferably, the nucleic acid sequence encodes at least one factor comprising both anti- angiogenic and neurotrophic properties. Most preferably, the factor comprising both anti- angiogenic and neurotrophic properties is pigment epithelium-derived factor (PEDF).
  • PEDF also named early population doubling factor- 1 (EPC-1)
  • serpins is a secreted protein having homology to a family of serine protease inhibitors named serpins.
  • PEDF is made predominantly by retinal pigment epithelial cells and is detectable in most tissues and cell types of the body. PEDF has been observed to induce differentiation in retinoblastoma cells and enhance survival of neuronal populations (Chader, Cell Different., 20, 209-216 (1987)). Factors that enhance neuronal survival under adverse conditions, such as PEDF, are termed "neuronotrophic," as described herein. PEDF further has gliastatic activity, or has the ability to inhibit glial cell growth. As discussed above, PEDF also has anti- angiogenic activity. Anti-angiogenic derivatives of PEDF include SLED proteins, discussed in WO 99/04806.
  • PEDF is involved with cell senescence (Pignolo et al., J Biol. Chem., 268(12), 8949-8957 (1998)).
  • PEDF for use in. the present inventive method can be derived from any source, and is further characterized in U.S. Patent 5,840,686 and International Patent Applications WO 93/24529 and WO 99/04806.
  • the present invention further provides a viral vector comprising a nucleic acid sequence encoding PEDF or a therapeutic fragment thereof, wherein the nucleic acid sequence operably linked to regulatory sequences necessary for expression of PEDF or a therapeutic fragment thereof.
  • the nucleic acid sequence can be obtained from any source, e.g., isolated from nature, synthetically generated, isolated from a genetically engineered organism, and the like. Appropriate viral vectors and regulatory sequences are discussed herein. In nature, PEDF is almost solely generated in human fetus retinal cells.
  • the viral vector of the present invention can be used to create sufficient amounts of recombinant PEDF or can be used in methods of research or treatment, e.g., the present inventive method.
  • the expression vector e.g., the adenoviral or the adeno-associated viral vector, also can comprise a nucleic acid sequence encoding a therapeutic fragment of at least one inhibitor of angiogenesis or at least one neurotrophic factor.
  • any inhibitor of angiogenesis or neurotrophic factor e.g., PEDF
  • PEDF neurotrophic factor
  • therapeutic fragments i.e., those fragments having biological activity sufficient to, for example, inhibit angiogenesis or promote neuron survival
  • nucleic acid sequences comprising substitutions, deletions, or additions, but which encode a functioning inhibitor of angiogenesis or neurotrophic factor or a therapeutic fragment of any of the foregoing.
  • a fusion protein comprising an anti-angiogenic factor or neurotrophic factor or a therapeutic fragment thereof and for example, a moiety that stabilizes peptide conformation, also can be present in the expression vector.
  • a functioning inhibitor of angiogenesis or a therapeutic fragment thereof prevents or ameliorates neovascularization.
  • a functioning neurotrophic factor or a therapeutic fragment thereof desirably promotes neuronal cell differentiation, inhibits glial cell proliferation, and/or promotes neuronal cell survival.
  • complete prevention or amelioration of neovascularization is not required in order to realize a therapeutic effect.
  • complete induction of neuron survival or differentiation is not required in order to realize a benefit.
  • a modified therapeutic factor or a fragment thereof has neurotrophic and anti-angiogenic therapeutic activity using, for example, neuronal cell differentiation and survival assays (see, for example, U.S. Patent 5,840,686), the mouse ear model of neovascularization, or the rat hindlimb ischemia model.
  • the inhibitor of angiogenesis and/or the neurotrophic factor can be a factor that acts upon a receptor for an anti-angiogenic factor or a receptor for a neurotrophic factor, thereby resulting in the desired biological effect.
  • the expression vector can comprise a nucleic acid sequence encoding an antibody that binds and activates the PEDF receptor, which signals a series of intracellular events responsible for the biological activity of PEDF.
  • PEDF receptors see, for example, Alberdi et al., J. Biol. Chem., 274(44), 31605 (1999).
  • the present invention also contemplates the use of nucleic acid sequences encoding chimeric or fusion peptides in the present inventive method.
  • nucleic acid sequences encoding chimeric or fusion peptides in the present inventive method.
  • scientists have been able to generate fusion proteins that contain the combined amino acid sequence of two or more proteins.
  • the ordinarily skilled artisan can fuse the active domains of two or more factors to generate chimeric peptides with desired activity.
  • the chimeric peptide can comprise the entire amino acid sequences of two or more peptides or, alternatively, can be constructed to comprise portions of two or more peptides (e.g., 10, 20, 50, 75, 100, 400, 500, or more amino acid residues).
  • the chimeric peptide comprises anti-angiogenic and neurotrophic activity, which can be determined using routine methods.
  • the expression vector of the present inventive method comprises a nucleic acid sequence that encodes at least one inhibitor of angiogenesis and/or at least one neurotrophic factor. Therefore, the nucleic acid sequence can encode multiple, i.e., two, three, or more, inhibitors of angiogenesis. Likewise, the nucleic acid sequence can encode multiple, i.e., two, three, or more, neurotrophic factors. In a preferred embodiment, the nucleic acid sequence encodes PEDF and ciliary neurotrophic factor (CNTF). Also preferably, the nucleic acid sequence encodes at least one inhibitor of angiogenesis and at least one neurotrophic factor.
  • CNTF ciliary neurotrophic factor
  • multiple inhibitors of angiogenesis and/or multiple neurotrophic factors can be operably linked to different promoters.
  • different promoters have dissimilar levels and patterns of activity.
  • One of ordinary skill in the art will appreciate the freedom to dictate the expression of different coding sequences through the use of multiple promoters.
  • the multiple coding sequences can be operably linked to the same promoter to form a polycistronic element.
  • the polycistronic element is transcribed into a single mRNA molecule when transduced into the ocular cell. Translation of the mRNA molecule is initiated at each coding sequence, thereby producing the multiple, separate peptides simultaneously.
  • the present invention also contemplates contacting an ocular cell or a cell associated with age-related macular degeneration with a cocktail of expression vectors, wherein each expression vector encodes a different inhibitor of angiogenesis and/or neurotrophic factor.
  • the cocktail of expression vectors can further comprise different types of expression vectors, e.g., adenoviral vectors and adeno-associated viral vectors.
  • the methods of the present invention can be part of a treatment regimen involving other therapeutic modalities. It is appropriate, therefore, if the ocular-related disorder, namely ocular neovascularization or age-related macular degeneration, has been treated, is being treated, or will be treated with any of a number of ocular therapies, such as drug therapy, photodynamic therapy, photocoagulation laser therapy, panretinal therapy, thermotherapy, radiation therapy, or surgery.
  • the surgery is macular translocation, removal of subretinal blood, or removal of subretinal choroidal neovascular membrane.
  • the expression vector is preferably administered intraocularly for the prophylactic or therapeutic treatment of age-related macular degeneration or persistent or recurrent ocular neovascularization treated with surgery, laser photocoagulation, and photodynamic therapies.
  • the expression vector is preferably administered as soon as possible after it has been determined that an animal, such as a mammal, specifically a human, is at risk for ocular neovascularization or age-related macular degeneration (prophylactic treatment) or has begun to develop ocular neovascularization or age-related macular degeneration (therapeutic treatment).
  • Treatment will depend, in part, upon the particular nucleic acid sequence used, the particular inhibitor of angiogenesis and/or neurotrophic factor expressed from the nucleic acid sequence, the route of administration, and the cause and extent, if any, of ocular neovascularization or age-related macular degeneration realized.
  • systemic administration or administration to both eyes is preferred in the prophylactic treatment of macular degeneration because, once one eye is affected, the other eye is at risk (up to 19% per year).
  • the expression vector of the present invention desirably is administered in a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and the expression vector(s).
  • a pharmaceutical composition which comprises a pharmaceutically acceptable carrier and the expression vector(s).
  • Any suitable pharmaceutically acceptable carrier can be used within the context of the present invention, and such carriers are well known in the art.
  • the choice of carrier will be determined, in part, by the particular site to which the composition is to be administered and the particular method used to administer the composition.
  • Suitable formulations include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood or intraocular fluid of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
  • Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the pharmaceutically acceptable carrier is a buffered saline solution.
  • the expression vector for use in the present inventive methods is administered in a pharmaceutical composition formulated to protect the expression vector from damage prior to administration.
  • the pharmaceutical composition can be formulated to reduce loss of the expression vector on devices used to prepare, store, or administer the expression vector, such as glassware, syringes, or needles.
  • the pharmaceutical composition can be formulated to decrease the light sensitivity and/or temperature sensitivity of the expression vector.
  • the pharmaceutical composition preferably comprises a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.
  • a pharmaceutical composition also can be formulated to enhance transduction efficiency.
  • the expression vector, e.g., viral vector, of the present invention can be present in a composition with other therapeutic or biologically-active agents. For example, therapeutic factors useful in the treatment of a particular indication can be present.
  • hyaluronidase can be added to a composition to effect the break down of blood and blood proteins in the vitreous of the eye.
  • Factors that control inflammation such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the viral vector and ocular distress.
  • Immune system suppressors can be administered in combination with the present inventive method to reduce any immune response to the vector itself or associated with an ocular disorder.
  • Anti-angiogenic factors such as soluble growth factor receptors, growth factor antagonists, i.e., angiotensin, and the like also can be part of the composition, as well as additional neurotrophic factors.
  • vitamins and minerals, anti-oxidants, and micronutrients can be co-administered.
  • Antibiotics i.e., microbicides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders.
  • suitable methods i.e., invasive and noninvasive methods, of administering an expression vector whereon the expression vector will contact an ocular cell are available.
  • invasive and noninvasive methods of administering an expression vector whereon the expression vector will contact an ocular cell are available.
  • a particular route can provide a more immediate and more effective reaction than another route. Accordingly, the described routes of administration are merely exemplary and are in no way limiting.
  • the present inventive methods are not dependent on the mode of administering the expression vector to an animal, preferably a human, to achieve the desired effect. As such, any route of administration is appropriate so long as the expression vector contacts an appropriate ocular cell.
  • the expression vector for use in the present inventive methods can be appropriately formulated and administered in the form of an injection, eye lotion, ointment, implant and the like.
  • the expression vector can be applied, for example, systemically, topically, subconjunctivally, intraocularly, retrobulbarly, periocularly, subretinally, or suprachoroidally.
  • Topical formulations are well known to those of skill in the art. Such formulations are suitable in the context of the present invention for application to the skin.
  • the expression vector can also be administered non-invasively using a needleless injection device, such as the Biojector 2000 Needle-Free Injection Management System® available from Bioject, Inc.
  • the expression vector is preferably present in or on a device that allows controlled or sustained release of the expression vector, such as an ocular sponge, meshwork, mechanical reservoir, or mechanical implant.
  • a device that allows controlled or sustained release of the expression vector such as an ocular sponge, meshwork, mechanical reservoir, or mechanical implant.
  • Implants see, e.g., U.S. Patents 5,443,505, 4,853,224 and 4,997,652
  • devices see, e.g., U.S. Patents 5,554,187, 4,863,457, 5,098,443 and 5,725,493
  • an implantable device e.g., a mechanical reservoir, an intraocular device or an extraocular device with an intraocular conduit, or an implant or a device comprised of a polymeric composition are particularly useful for ocular administration of the expression vector.
  • the expression vector of the present inventive methods can also be administered in the form of sustained-release formulations (see, e.g., U.S. Patent 5,378,475) comprising, for example, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), or a polylactic- glycolic acid.
  • sustained-release formulations see, e.g., U.S. Patent 5,378,475
  • chondroitin sulfate a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), or a polylactic- glycolic acid.
  • BHET bis-2-hydroxyethyl-terephthalate
  • the expression vector can be administered using invasive procedures, such as, for instance, intravitreal injection or subretinal injection optionally preceded by a vitrectomy.
  • Subretinal injections can be administered to different compartments of the eye, i.e., the anterior chamber.
  • injectable compositions can also be administered intramuscularly, intravenously, and intraperitoneally.
  • Pharmaceutically acceptable carriers for injectable compositions are well-known to those of ordinary skill in the art (see Pharmaceutics and Pharmacy Practice, J.B.
  • the expression vector can also be administered in vivo by particle bombardment, i.e., a gene gun.
  • the expression vector is administered via an ophthalmologic instrument for delivery to a specific region of an eye.
  • an ophthalmologic instrument ensures precise administration of the expression vector while minimizing damage to adjacent ocular tissue. Delivery of the expression vector to a specific region of the eye also limits exposure of unaffected cells to the inhibitor of angiogenesis and/or neurotrophic factor, thereby reducing the risk of side effects.
  • a preferred ophthalmologic instrument is a combination of forceps and subretinal needle or sharp bent cannula.
  • the expression vector can be administered parenterally.
  • any expression vector parenterally administered to a patient for the prophylactic or therapeutic treatment of an ocular-related disorder i.e., ocular neovascularization or age-related macular degeneration
  • an expression vector can be modified to alter the binding specificity or recognition of an expression vector for a receptor on a potential host cell.
  • such manipulations can include deletion of regions of the fiber, penton, or hexon, insertions of various native or non-native ligands into portions of the coat protein, and the like.
  • parenteral administration can require large doses or multiple administrations to effectively deliver the expression vector to the appropriate host cells.
  • dosage and routes of administration can be selected to minimize loss of expression vector due to a host's immune system.
  • a null expression vector i.e., an expression vector not comprising the nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic factor
  • Prior administration of null expression vectors can serve to create an immunity in the host to the expression vector, thereby decreasing the amount of vector cleared by the immune system.
  • the dose of expression vector administered to an animal, particularly a human, in accordance with the present invention should be sufficient to effect the desired response in the animal over a reasonable time frame.
  • dosage will depend upon a variety of factors, including the age, species, the pathology in question, and condition or disease state. Dosage also depends on the inhibitor of angiogenesis and/or neurotrophic factor to be expressed, as well as the amount of ocular tissue about to be affected or actually affected by the ocular-related disease.
  • the size of the dose also will be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular expression vector and the desired physiological effect. It will be appreciated by one of ordinary skill in the art that various conditions or disease states, in particular, chronic conditions or disease states, may require prolonged treatment involving multiple administrations.
  • Suitable doses and dosage regimens can be determined by conventional range- finding techniques known to those of ordinary skill in the art.
  • the about 10 6 viral particles to about 10 12 viral particles are delivered to the patient.
  • a pharmaceutical composition can be administered that comprises an expression vector concentration of from about 10 6 particles/ml to about 10 12 particles/ml (including all integers within the range of about 10 particles/ml to about 10 particles/ml), preferably from about 10 10 particles/ml to about 10 12 particles/ml, and will typically involve the intraocular administration of from about 0.1 ⁇ l to about 100 ⁇ l of such a pharmaceutical composition per eye.
  • the expression vector is a plasmid, preferably about 0.5 ng to about 1000 ⁇ g of DNA is administered.
  • 0.1 ⁇ g to about 500 ⁇ g is administered, even more preferably about 1 ⁇ g to about 100 ⁇ g of DNA is administered. Most preferably, about 50 ⁇ g of DNA is administered per eye.
  • other routes of administration may require smaller or larger doses to achieve a therapeutic effect. Any necessary variations in dosages and routes of administration can be determined by the ordinarily skilled artisan using routine techniques known in the art.
  • the present inventive method provides for multiple applications of the inhibitor of angiogenesis and/or neurotrophic agent to prophylactically or therapeutically treat ocular neovascularization or other ocular-related disorders.
  • at least two applications of an expression vector comprising an exogenous nucleic acid e.g., a nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic agent
  • the multiple doses are administered while retaining gene expression above background levels.
  • the ocular cell is contacted with two applications or more of the expression vector within about 30 days or more. More preferably, two or more applications are administered to ocular cells of the same eye within about 90 days or more.
  • an expression vector comprising a nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic factor can be introduced ex vivo into cells, preferably ocular cells, previously removed from a given animal, in particular a human.
  • Such transduced autologous or homologous host cells reintroduced into the animal or human, will express directly at least one inhibitor of angiogenesis and/or at least one neurotrophic factor in vivo.
  • One ex vivo therapeutic option involves the encapsidation of infected ocular cells into a biocompatible capsule, which can be implanted in the eye or any other part of the body.
  • the expression vector preferably the adenoviral vector, can comprise multiple nucleic acid sequences encoding the at least one inhibitor of angiogenesis and/or the at least one neurotrophic factor.
  • the expression vector can comprise multiple copies of the PEDF coding sequence, each copy operably linked to a different promoter or to identical promoters.
  • the nucleic acid sequence encoding at least one inhibitor of angiogenesis and/or at least one neurotrophic factor can further comprise one or more other transgenes.
  • transgene is meant any nucleic acid that can be expressed in a cell.
  • the expression of the transgene is beneficial, e.g., prophylactically or therapeutically beneficial, to the ocular cell or eye. If the transgene confers a prophylactic or therapeutic benefit to the cell, the transgene can exert its effect at the level of RNA or * protein.
  • the transgene can encode a peptide other than an inhibitor of angiogenesis or neurotrophic factor that can be employed in the treatment or study of a disorder, e.g., an ocular-related disorder.
  • the transgene can encode an antisense molecule, a ribozyme, a protein that affects splicing or 3' processing (e.g., polyadenylation), or a protein that affects the level of expression of another gene within the cell (i.e., where gene expression is broadly considered to include all steps from initiation of transcription through production of a process protein), such as by mediating an altered rate of mRNA accumulation or transport or an alteration in post-transcriptional regulation.
  • the transgene can encode a chimeric peptide for combination treatment of an ocular-related disorder.
  • the transgene can encode a factor that acts upon a different target molecule than the inhibitor of angiogenesis or the neurotrophic agent.
  • the transgene product can act upon a different signal transduction pathway, or can act at different points of the same signal transduction pathway of the inhibitor of angiogenesis or the neurotrophic factor.
  • the therapeutic substance is a neurotrophic factor, such as CNTF.
  • CNTF belongs to the neuropoietic cytokines subclass of neurotrophic factors. CNTF promotes the survival of ciliary ganglionic neurons and supports certain neurons that are nerve growth factor (NGF)-responsive.
  • NGF nerve growth factor
  • one or more additional nucleic acid sequences that encode a factor associated with cell differentiation can be included in the expression vector (e.g., viral vector).
  • the transgene encodes an atonal-associated peptide such as Mathl or Hathl or a biologically active fragment of either of the foregoing.
  • Mathl is a member of the mouse basic helix-loop-helix family of transcription factors and is homologous to the Drosophila gene atonal.
  • Hathl is the human counterpart of Mathl .
  • Mathl has been shown to be essential for hair development and can stimulate hair regeneration in the ear.
  • the expression vector can comprise a nucleic acid sequence encoding a vessel maturation factor in addition to at least one inhibitor of angiogenesis and/or at least one neurotrophic factor.
  • vessel maturation factors reduce the amount of vascular leakage and, therefore, are useful in treating, for example, exudative ocular disorders.
  • Vessel maturation factors include, but are not limited to, angiopoietins (Ang, e.g., Ang-1 and Ang-2), tumor necrosis factor-alpha (TNF- ⁇ ), midkine (MK), COUP-TFII, and heparin-binding neurotrophic factor (HBNF, also known as heparin binding growth factor).
  • Ang angiopoietins
  • TNF- ⁇ tumor necrosis factor-alpha
  • MK midkine
  • COUP-TFII heparin-binding neurotrophic factor
  • HBNF heparin-binding neurotrophic factor
  • the expression vector can comprise one or more additional nucleic acid sequences encoding an additional anti-angiogenic substance.
  • an anti- angiogenic substance is any biological factor that prevents or ameliorates neovascularization.
  • an anti-angiogenic substance can effect partial or complete prevention and amelioration of angiogenesis to achieve a therapeutic effect.
  • An anti-angiogenic substance includes, for instance, an anti- angiogenic factor, an anti-sense molecule specific for an angiogenic factor, a ribozyme, a receptor for an angiogenic factor, and an antibody that binds a receptor for an angiogenic factor.
  • the transgene can encode a marker protein, such as green fluorescent protein or luciferase. Such marker proteins are useful in vector construction and determining vector migration. Marker proteins also can be used to determine points of injection or treated ocular tissues in order to efficiently space injections of the expression vector to provide a widespread area of treatment, if desired. Alternatively, the transgene can encode a selection factor, which also is useful in vector construction protocols. If desired, the transgene can be part of an expression cassette.
  • a marker protein such as green fluorescent protein or luciferase.
  • marker proteins are useful in vector construction and determining vector migration. Marker proteins also can be used to determine points of injection or treated ocular tissues in order to efficiently space injections of the expression vector to provide a widespread area of treatment, if desired.
  • the transgene can encode a selection factor, which also is useful in vector construction protocols. If desired, the transgene can be part of an expression cassette.
  • any of the nucleic acid sequences described herein can be altered from their native form to increase their therapeutic effect.
  • a cytoplasmic form of a therapeutic nucleic acid can be converted to a secreted form by incorporating a signal peptide into the encoded gene product.
  • the at least one inhibitor of angiogenesis and/or at least one neurotrophic factor can be designed to be taken up by neighboring cells by fusion of the peptide with VP22. This allows an ocular cell comprising the therapeutic nucleic acid to have a therapeutic effect on a number of ocular cells within the mammal.
  • the present inventive methods also can involve the co-administration of other pharmaceutically active compounds.
  • co-administration is meant administration before, concurrently with, e.g., in combination with the expression vector in the same formulation or in separate formulations, or after administration of the expression vector as described above.
  • factors that control inflammation such as ibuprofen or steroids, can be co-administered to reduce swelling and inflammation associated with intraocular administration of the expression vector.
  • Immunosuppressive agents can be co- administered to reduce inappropriate immune responses related to an ocular disorder or the practice of the present inventive method.
  • Anti-angiogenic factors such as soluble growth factor receptors, growth factor antagonists, i.e., angiotensin, and the like can also be co- administered, as well as neurotrophic factors.
  • vitamins and minerals, anti- oxidants, and micronutrients can be co-administered.
  • Antibiotics i.e., microbicides and fungicides, can be co-administered to reduce the risk of infection associated with ocular procedures and some ocular-related disorders.
  • the expression vectors of the instant invention are particularly useful in the study or treatment of ocular disorders, including ocular disorders comprising a neovascular component and age-related macular degeneration, it will be appreciated that the expression vectors can be used to research and/or treat prophylactically or therapeutically a wide variety of animal diseases.
  • the viral vector comprising a nucleic acid sequence encoding PEDF or a therapeutic fragment thereof can be used in the study or treatment of the nervous system, genitourinary ailments, cancer, infectious disease, and cardiovascular abnormalities, as well as miscellaneous other health nuisances.
  • the viral vector can be used to study or treat, for example, sleep disorders, ALS (Lou Gehrig's Disease), Alzheimer's Disease, epilepsy, multiple sclerosis, Parkinson's Disease, peripheral neuropathies, Schizophrenia, depression, anxiety, spinal cord injury, traumatic brain injury, or acute, chronic, or inflammatory pain.
  • the expression vectors of the present invention can be used to treat genitourinary ailments, which include, for example, benign prostatic hyperplasia (BPH), impotence, neurogenic bladder, urinary incontinence, kidney failure, and end stage renal disease.
  • BPH benign prostatic hyperplasia
  • the expression vectors are useful in treating cancer such as, for example, cancer of the bladder, brain, breast, colorectal, esophageal, head & neck, liver/hepatoma, lung, melanoma, ovarian, pancreatic, prostate, stomach, testicular, uterine/endometrial, leukemias, and lymphomas.
  • Exemplary infectious diseases for treatment with the expression vectors include, but are not limited to, chlamydia, herpes, malaria, human papilloma virus (HPV), AIDS/HIV, pneumococcal pneumonia, influenza, meningitis, hepatitis, and tuberculosis.
  • Cardiovascular diseases such as, for example, neovascular diseases, ischemia, congestive heart failure, coronary artery disease, arrhythmia, atherosclerosis, increased LDL/HDL ratios, restenosis after angioplasty or in- stent restenosis, stroke, sickle cell anemia, and hemophilia, can be treated or studied, as well as the alleviation of, for example, obesity, organ transplantation/transplant rejection, osteoporosis, alopecia, hair loss, arthritis, allergies (such as to ragweed, pollen, and animal dander), cystic fibrosis, diabetes, and hearing loss.
  • neovascular diseases such as, for example, neovascular diseases, ischemia, congestive heart failure, coronary artery disease, arrhythmia, atherosclerosis, increased LDL/HDL ratios, restenosis after angioplasty or in- stent restenosis, stroke, sickle cell anemia, and hemophilia
  • neovascular diseases
  • This example illustrates a preferred method of obtaining expression of a factor comprising both anti-angiogenic and neurotrophic activity from an adenoviral vector in in vivo retina.
  • E3, and E4 regions of the adenoviral genome and comprising a PEDF gene is preferably constructed as set forth in WO 99/15686 (McVey et al.).
  • the method of the present invention is not dependent on the method of vector construction employed and previously described methods of vector construction are also suitable.
  • Several in vivo models of ocular neovascularization are available. Neovascularization of the retina is obtained in, for example, neonatal animals, i.e., neonatal mice, by exposing the mice to hypoxic conditions shortly after birth. Several days later, the neonatal mice are exposed to standard atmospheric conditions, resulting in ischemia-induced neovascularization of the retina.
  • Ad. PEDF is administered to the right eye of at least 12 day old mice anesthetized with, for example, ketamine or a combination of ketamine and xylazine via intravitreal injection. Injections are performed by forming an entrance site in the posterior portion of the eye and administering approximately 0.1-5.0 ⁇ l of composition comprising Ad.PEDF. In most instances, an injection of the expression vector will be administered to only one eye, while the remaining eye serves as a control. The mice are sacrificed at various time points after administration to determine the extent and duration of PEDF expression in the retina. The right and left eyes of each animal are enucleated and either fixed for histological analysis or prepared for PEDF expression analysis.
  • PEDF DNA, PEDF RNA, or PEDF protein can be accomplished using methods well known in the art, such as PCR and blotting techniques (see, for example, Sambrook et al., supra).
  • PCR and blotting techniques See, for example, Sambrook et al., supra.
  • This example demonstrates a preferred method of obtaining expression of a factor comprising both anti-angiogenic and neurotrophic activity from an adenoviral vector in in vivo choroid.
  • the following example further provides methods for determining the effect of PEDF on neovascularization.
  • E3, and E4 regions of the adenoviral genome and comprising a PEDF gene is constructed as set forth in WO 99/15686 (McVey et al.).
  • An in vivo model of choroidal neovascularization can be obtained by detaching the retina of an eye of, for example, a mouse or rabbit, and debriding the pigmented epithelia.
  • Ad.PEDF is administered prior to perturbing the retinal pigment epithelial (RPE) to determine the effect of the present inventive method in preventing choroidal neovascularization.
  • Ad.PEDF is administered after perturbing the retina and RPE for determining the therapeutic effect of the procedure on neovascularization.
  • Choroidal neovascularization can be monitored in vivo using fundus photography, fluorescein angiography and/or indocyanine-green angiography, as commonly used in the art. Using these methods, one of ordinary skill in the art is able to detect growth of new blood vessels and vascular leakage often associated with neovascularization. For research purposes, neovascularization can also be determined by enucleating the eyes and preparing vascular casts or examining ocular tissue via scanning electron microscopy.
  • This example demonstrates the utility of adenoviral vectors to deliver multiple doses of an exogenous nucleic acid to the eye.
  • Adenoviral vectors comprising the luciferase gene (Ad.L) or control adenoviral vectors comprising no transgene (Ad.null) were injected into the intravitreal space of C57BL6 mouse eyes (Day 0).
  • Ad.L luciferase gene
  • Ad.null control adenoviral vectors comprising no transgene
  • Group I eyes were enucleated and frozen the day following the second administration of adenoviral vectors (Day 15, 2 nd administration).
  • Group II eyes were injected intravitreally with Ad.null at Day 14, and injected intravitreally with Ad.L vectors four weeks following the initial injection with Ad.null (Day 28, 3 rd administration). The eyes were then enucleated and frozen the day after the third administration of adenoviral vector.
  • Group III eyes were injected intravitreally with Ad.null at Day 14 and Day 28, and injected with Ad.L vectors six weeks following the initial injection with Ad.null (Day 42, 4 administration). The eyes were then enucleated and frozen the day after the fourth administration of adenoviral vector.
  • Luciferase assays were performed on the eye samples to determine the efficiency of infection and gene expression associated with multiple dosing of the vectors.
  • Luciferase expression in ocular cells after the 1 st and 2 nd administration of adenoviral vector was substantially equivalent. In other words, no loss of gene expression was detected following two administrations of the gene transfer vector. Gene expression from the 3 rd administration of adenoviral vector was between 10- and 100-fold reduced compared to gene expression from the 1 st administration and the 2 nd administration, but was still above background levels (e.g., as detected in cells transduced with Ad.null). Gene expression from the 4 th administration of adenoviral vector was reduced approximately 3- to 10-fold compared to the gene expression observed following the 3 rd administration. However, the level of gene expression following the 4 th administration was above background levels.
  • This example demonstrates the feasibility of performing multiple applications of adenoviral vectors to the eye in order to obtain expression of an exogenous nucleic acid in ocular cells.
  • This example demonstrates the ability of an expression vector comprising a nucleic acid sequence encoding a factor comprising both anti-angiogenic and neurotrophic properties to inhibit choroidal neovascularization (CNV).
  • CNV choroidal neovascularization
  • AdPEDF.10 Replication-deficient (El -/E3 -deficient) adenoviral vectors (AdPEDF.10) comprising the coding sequence for PEDF operably linked to the CMN immediate early promoter were constructed using standard techniques. A null version of the vector (Ad ⁇ ull.10), which did not comprise the PEDF coding sequence, was also constructed.
  • Ad ⁇ ull.10 Replication-deficient (El -/E3 -deficient) adenoviral vectors
  • AdPEDF.10 using a Harvard pump microinjection apparatus and pulled glass micropipettes. Each eye was injected intravitreously with 1 ⁇ l of vehicle containing 10 particles of virus. Alternatively, each eye was injected subretinally with 10 particles of virus suspended in 1 ⁇ l of vehicle. Five days post-injection, mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight). Topicamide (1%) was utilized to dilate the pupils prior to rupture of Bruch's membrane by diode laser photocoagulation. Rupture of Bruch's membrane is known to induce neovascularization of the choroid.
  • choroidal flat mounts (described in Ed ⁇ lman et al., Invest. Ophthalmol. Vis. Sci., 41, S834 (2000)) were prepared to observe the degree of neovascularization of the choroidal membrane. Briefly, eyes were removed from the subjects and fixed in phosphate-buffered formalin. The cornea, lens, and retina were removed from the eyecup, and the eyecup was flat-mounted. Flat mounts were then examined by fluorescence microscopy and images were digitized using a 3 color CCD video camera (IK-TU40A, Toshiba, Tokyo, Japan) for computer image analysis.
  • IK-TU40A 3 color CCD video camera
  • This example demonstrates the ability of an expression vector comprising a nucleic acid sequence encoding a factor comprising both anti-angiogenic and neurotrophic properties to inhibit ischemia-induced retinal neovascularization.
  • Replication-deficient adenoviral vectors comprising the coding sequence for PEDF operably linked to the CMN immediate early promoter were constructed using standard techniques.
  • Ischemic retinopathy was produced in adult C57BL/6 mice as previously described (see, for example, Smith et al., Invest. Ophthalmol. Vis. Sci, 35, 101 (1994)). Briefly, seven day old mice (P7) were exposed to an atmosphere of 75 +/- 3% oxygen for five days. At P10, mice were injected intravitreously with 10 9 particles of AdPEDF.11 or AdNull.11 , returned to oxygen for two days, then returned to room atmosphere. At P 17, the mice were sacrificed and eyes were rapidly removed and frozen in optimum cutting temperature embedding compound (OCT; Miles Diagnostics, Elkhart, IN).
  • OCT cutting temperature embedding compound

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Abstract

La présente invention concerne un procédé destiné au traitement prophylactique ou thérapeutique d'un animal pour au moins un trouble oculaire, comme par exemple, une néovascularisation oculaire ou une dégénérescence maculaire liée à l'âge. Ce procédé consiste à mettre une cellule oculaire en contact avec un vecteur d'expression comprenant une séquence d'acide nucléique codant un inhibiteur de l'angiogénèse et la même séquence d'acide nucléique ou une séquence différente codant un agent neurotrophique. Ce procédé consiste également à mettre une cellule oculaire en contact avec différents vecteurs d'expression, chacun comprenant une séquence d'acide nucléique codant un inhibiteur de l'angiogénèse et/ou une séquence d'acide nucléique codant un agent neurotrophique. De plus, la présente invention concerne un vecteur viral comprenant une séquence d'acide nucléique codant le pigment epithelium-derived factor (PEDF) ou un fragment thérapeutique de celui-ci.
PCT/US2001/004203 2000-02-11 2001-02-09 Matieres et procedes destines au traitement de troubles oculaires WO2001058494A2 (fr)

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JP2001557602A JP2003522160A (ja) 2000-02-11 2001-02-09 眼関連疾患の治療のための遺伝子治療
AU36818/01A AU780634C (en) 2000-02-11 2001-02-09 Gene Therapy for treating ocular-related disorders
US10/211,701 US20030045498A1 (en) 2000-02-11 2002-08-02 Materials and methods for treating ocular-related disorders
AU2005202935A AU2005202935A1 (en) 2000-02-11 2005-07-06 Gene therapy for treating ocular-related disorders
US11/610,364 US20070098692A1 (en) 2000-02-11 2006-12-13 Materials and methods for treating ocular-related disorders
US12/237,036 US20090018100A1 (en) 2000-02-11 2008-09-24 Materials and methods for treating ocular-related disorders

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WO2004076626A3 (fr) * 2003-02-24 2004-10-28 Genvec Inc Materiaux et procedes de traitement des troubles de l'audition
WO2004050027A3 (fr) * 2002-12-02 2004-12-02 Genvec Inc Procedes et materiaux destines a traiter des troubles oculaires
US7470659B2 (en) 2001-12-07 2008-12-30 The Regents Of The University Of California Methods to increase reverse cholesterol transport in the retinal pigment epithelium (RPE) and Bruch's membrane (BM)
WO2009126894A2 (fr) * 2008-04-11 2009-10-15 Massachusetts Eye And Ear Infirmary Procédés et compositions pour le diagnostic et le traitement de troubles angiogènes
US8173591B2 (en) 2004-11-16 2012-05-08 Yeda Research And Development Co. Variants of pigment epithelium derived factor and uses thereof
WO2014159546A1 (fr) * 2013-03-14 2014-10-02 Asklepios Biopharmaceutical, Inc. Gènes de recepteur-1 vegf soluble modifié et vecteurs pour une thérapie génique
US9951351B2 (en) 2014-10-09 2018-04-24 Genvec, Inc. Adenoviral vector encoding human atonal homolog-1 (HATH1)
WO2019246130A1 (fr) * 2018-06-19 2019-12-26 Cella Therapeutics, Llc Systèmes d'administration de médicament à libération prolongée comprenant un agent abaissant la pression intraoculaire, un composé de cnp, un composé de npr-b, un agoniste de tie-2, ou un agent neurotrophique destinés à être utilisés pour traiter le glaucome ou l'hypertension oculaire
WO2019246141A1 (fr) * 2018-06-19 2019-12-26 Cella Therapeutics, Llc SYSTÈMES D'ADMINISTRATION DE MÉDICAMENT COMPRENANT UN AGENT NEUROTROPHIQUE, UN INHIBITEUR DE FRAGMENT DE SIGNALISATION D'APOPTOSE (FAS) OU UN INHIBITEUR DE LIGAND DE FAS (FASL), UN INHIBITEUR DU FACTEUR DE NÉCROSE TUMORALE-α OU DU RÉCEPTEUR DU TNF, UN PEPTIDE MITOCHONDRIAL, UN OLIGONUCLÉOTIDE, INHIBITEUR DE CHIMIOKINE, UNE CYSTÉINE-PROTÉASE ASPARTIQUE
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WO2019246141A1 (fr) * 2018-06-19 2019-12-26 Cella Therapeutics, Llc SYSTÈMES D'ADMINISTRATION DE MÉDICAMENT COMPRENANT UN AGENT NEUROTROPHIQUE, UN INHIBITEUR DE FRAGMENT DE SIGNALISATION D'APOPTOSE (FAS) OU UN INHIBITEUR DE LIGAND DE FAS (FASL), UN INHIBITEUR DU FACTEUR DE NÉCROSE TUMORALE-α OU DU RÉCEPTEUR DU TNF, UN PEPTIDE MITOCHONDRIAL, UN OLIGONUCLÉOTIDE, INHIBITEUR DE CHIMIOKINE, UNE CYSTÉINE-PROTÉASE ASPARTIQUE
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WO2019246130A1 (fr) * 2018-06-19 2019-12-26 Cella Therapeutics, Llc Systèmes d'administration de médicament à libération prolongée comprenant un agent abaissant la pression intraoculaire, un composé de cnp, un composé de npr-b, un agoniste de tie-2, ou un agent neurotrophique destinés à être utilisés pour traiter le glaucome ou l'hypertension oculaire
US12128104B2 (en) 2018-06-19 2024-10-29 Cella Therapeutics, Llc Drug delivery systems comprising a neurotrophic agent, an apoptosis signaling fragment inhibitor (FAS) or FAS ligand (FASL) inhibitor, a tumor necrosis factor-alpha (TNF-alpha) or TNF receptor inhibitor, a mitochondrial peptide, an oligonucleotide, a chemokine inhibitor, or a cysteine-aspartic protease inhibitor
AU2022247731B2 (en) * 2018-06-19 2025-01-23 Cella Therapeutics, Llc Drug delivery systems comprising a neurotrophic agent, an apoptosis signaling fragment inhibitor (FAS) or FAS ligand (FASL) inhibitor, a tumor necrosis factor-alpha (TNF-alpha) or TNF receptor inhibitor, a mitochondrial peptide, an oligonucleotide, a chemokine inhibitor, or a cysteine-aspartic protease
WO2021127052A1 (fr) * 2019-12-18 2021-06-24 Cella Therapeutics, Llc Systèmes d'administration de médicament comprenant un agent neurotrophique, un inhibiteur de fragment de signalisation d'apoptose (fas) ou un inhibiteur de ligand de fas (fasl), un inhibiteur du facteur de nécrose tumorale α (tnf-a) ou du récepteur du tnf, un peptide mitochondrial, un oligonucléotide, un inhibiteur de chimiokine ou un inhibiteur de protéase aspartique à cystéine

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AU3681801A (en) 2001-08-20
CA2398571A1 (fr) 2001-08-16
EP1253948A2 (fr) 2002-11-06
WO2001058494A3 (fr) 2002-04-04
AU780634B2 (en) 2005-04-07
JP2003522160A (ja) 2003-07-22

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